Method for glycosylating hydroxylysine residues in polypeptides or peptides

Abstract

This invention relates to a multifunctional enzyme having lysyl hydroxylase and glycosyltransferase activity and to the use of the enzyme in glycosylating hydroxylysine residues. In particular this invention relates to a method for producing glycosyltransferase activity, which comprises that a nucleotide sequence encoding lysyl hydroxylase and glycosyltransferase activity is introduced and expressed in a chosen host and the protein product is recovered from the host cell or from the culture medium.

1. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising: glycosylating a hydroxylysine residue with a lysyl hydroxylase (LH) having at least 90% identity to an amino acid sequence having SEQ ID NO: 4 and having glycosyltransferase activity. 
     
     
       2. The method of  claim 1 , further comprising: expressing in a host a nucleotide sequence encoding lysyl hydroxylase enzyme having glycosyltransferase and lysyl hydroxylase activities. 
     
     
       3. The method according to  claim 1  or  2 , wherein the glycosylation is carried out in the presence of additives and factors necessary for the function of the glycosyltransferase enzyme. 
     
     
       4. The method according to  claim 1 , wherein the lysyl hydroxylase enzyme has glucosyltransferase activity and/or galactosyltransferase activity. 
     
     
       5. The method according to  claim 1 , wherein the enzyme is encoded by a nucleotide sequence originating from an organism comprising polypeptides or peptides having hydroxylysine residues. 
     
     
       6. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising:
 (1) expressing in a host a nucleotide sequence encoding an enzyme having lysyl hydroxylase and glycosyltransferase activities; and 
 (2) glycosylating a hydroxylysine residue with said enzyme, wherein said enzyme is selected from the group consisting of:
 (a) a polypeptide having the sequence set forth in SEQ ID NO: 4; 
 (b) a polypeptide comprising an amino acid sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 4; 
 (c) a polypeptide having the amino acid sequence of amino acids 1-660 of SEQ ID NO: 4 and having GGT activity; 
 (d) a polypeptide having the amino acid sequence of amino acids 1-507 of SEQ ID NO: 4 and having GGT activity; 
 (e) a polypeptide having at least 90% identity to the amino acid sequence of amino acids 1-660 of SEQ ID NO: 4 and having GGT activity, and 
 (f) a polypeptide having at least 90% identity to the amino acid sequence of amino acids 1-507 of SEQ ID NO: 4 and having GGT activity. 
 
 
     
     
       7. The method of  claim 1 , further comprising: expressing in a host a nucleotide sequence encoding nematode LH wherein said nematode LH does not have lysyl hydroxylase activity due to a genetic modification of SEQ ID NO: 3, wherein said genetic modification causes in the encoded polypeptide the amino acid changes selected from a group consisting of:
 (a) aspartate to alanine at amino acid position 661, and 
 (b) deletion of 70 to 223 amino acids from the carboxy terminus, 
 and wherein said nematode LH has glycosyltransferase activity. 
 
     
     
       8. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising: expressing in a host a protein encoded by a nucleotide sequence consisting of SEQ ID NO: 3, and glycosylating a hydroxylysine residue with said protein. 
     
     
       9. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising glycosylating a hydroxylysine residue with a lysyl hydroxylase enzyme, wherein the lysyl hydroxylase enzyme is encoded by a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 3, and 
 (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4. 
 
     
     
       10. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising glycosylating a hydroxylysine residue with a lysyl hydroxylase enzyme, wherein the lysyl hydroxylase enzyme comprises a polypeptide encoded by a nucleotide sequence that encodes a polypeptide having an amino acid sequence which shows at least 90% sequence identity to SEQ ID NO: 4. 
     
     
       11. The method of  claim 10  wherein said amino acid has the amino acid sequence of SEQ ID NO: 4 wherein the aspartic acid at position 661 is substituted with an alanine. 
     
     
       12. The method according to  claim 10 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed and optionally secreted in a host selected from the group comprising a bacterial host, a fungal host, a yeast host, an insect cell, a plant cell, an animal cell, a plant host, a non-human animal host and/or human host. 
     
     
       13. The method according to  claim 10 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in  E. coli.    
     
     
       14. The method according to  claim 10 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in an insect cell. 
     
     
       15. The method according to  claim 10 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in a mammalian host cell. 
     
     
       16. The method according to  claim 10 , wherein the nucleotide sequence is introduced and expressed in a chosen milk-producing animal and the protein product is recovered from the milk of the milk-producing animal. 
     
     
       17. The method according to  claim 10 , wherein the glycosyltransferase enzyme is in multimeric or monomeric form. 
     
     
       18. The method according to  claim 10 , wherein the hydroxylysine of collagen, collagenous protein, collagen-type protein or other protein or synthetic or partly synthetic protein is glycosylated. 
     
     
       19. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising glycosylating a hydroxylysine residue with a lysyl hydroxylase enzyme, wherein the lysyl hydroxylase enzyme comprises a polypeptide encoded by a nucleotide sequence encoding a polypeptide having the amino acid sequence of amino acids 1-507 of SEQ ID NO: 4. 
     
     
       20. A method for glycosylating hydroxylysine residues in polypeptides or peptides having these residues, the method comprising glycosylating a hydroxylysine residue with a lysyl hydroxylase enzyme, wherein the lysyl hydroxylase enzyme comprises a polypeptide sequence encoded by a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of amino acids 1-660 of SEQ ID NO: 4; 
 (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence of amino acids 1-507 of SEQ ID NO: 4; 
 (c) a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of amino acids 1-660 of SEQ ID NO: 4; and 
 (d) a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of amino acids 1-507 of SEQ ID NO: 4. 
 
     
     
       21. The method according to  claim 20 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed and optionally secreted in a host selected from the group comprising a bacterial host, a fungal host, a yeast host, an insect cell, a plant cell, an animal cell, a plant host, a non-human animal host and/or human host. 
     
     
       22. The method according to  claim 20 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in  E. coli.    
     
     
       23. The method according to  claim 20 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in an insect cell. 
     
     
       24. The method according to  claim 20 , wherein the nucleotide sequence encoding lysyl hydroxylase is expressed in a mammalian host cell. 
     
     
       25. The method according to  claim 20 , wherein the nucleotide sequence is introduced and expressed in a chosen milk-producing animal and the protein product is recovered from the milk of the milk-producing animal. 
     
     
       26. The method according to  claim 20 , wherein the glycosyltransferase enzyme is in multimeric or monomeric form. 
     
     
       27. The method according to  claim 20 , wherein the hydroxylysine of collagen, collagenous protein, collagen-type protein or other protein or synthetic or partly synthetic protein is glycosylated.